The cyanobacterium Synechocystis sp. PCC 6803 contains four members of the FtsH protease family. One of these, FtsH (slr0228), has been implicated recently in the repair of photodamaged photosystem II (PSII) complexes. We have demonstrated here, using a combination of blue native PAGE, radiolabeling, and immunoblotting, that FtsH (slr0228) is required for selective replacement of the D1 reaction center subunit in both wild type PSII complexes and in PSII subcomplexes lacking the PSII chlorophyll a-binding subunit CP43. To test whether FtsH (slr0228) has a more general role in protein quality control in vivo, we have studied the synthesis and degradation of PSII subunits in wild type and in defined insertion and missense mutants incapable of proper assembly of the PSII holoenzyme. We discovered that, when the gene encoding FtsH (slr0228) was disrupted in these strains, the overall level of assembly intermediates and unassembled PSII proteins markedly increased. Pulse-chase experiments showed that this was due to reduced rates of degradation in vivo. Importantly, analysis of epitope-tagged and green fluorescent protein-tagged strains revealed that slr0228 was present in the thylakoid and not the cytoplasmic membrane. Overall, our results show that FtsH (slr0228) plays an important role in controlling the removal of PSII subunits from the thylakoid membrane and is not restricted to selective D1 turnover.All cellular organisms possess quality control mechanisms to prevent the accumulation of unwanted proteins (1). Of current interest are the processes by which damaged and unassembled proteins are removed from the thylakoid membrane, which is the location of the protein complexes involved in oxygenic photosynthetic electron transport in cyanobacteria and chloroplasts (2). Side reactions associated with the light reactions of photosynthesis lead to the production of a variety of potentially hazardous molecules (e.g. reactive oxygen species) that can oxidize components, such as amino acid side chains and pigment cofactors (3). Ultimately, unless the damage is repaired, there is a net reduction in photosynthetic performance known as photoinhibition (for review see Ref. 4). As yet, the repair pathways and, more generally, the quality control processes involved in the assembly of the thylakoid membrane protein complexes and removal of aberrant proteins remain poorly understood.One component that is especially prone to photodamage is the photosystem II (PSII) 2 complex, which is composed of over 25 membrane and peripheral proteins and is responsible for the light-driven oxidation of water and reduction of plastoquinone (5). The D1 protein, together with the homologous D2 protein, binds the cofactors involved in electron transfer through the complex (6). The D1 subunit is also the chief target for light-induced damage in PSII and undergoes rapid synthesis and degradation in the light to maintain PSII activity in what is termed the PSII repair cycle (4).Although the precise mechanism of PSII repair is unknown, recent work ha...

FtsH metalloproteases are key components of the photosystem II (PSII) repair cycle, which operates to maintain photosynthetic activity in the light. Despite their physiological importance, the structure and subunit composition of thylakoid FtsH complexes remain uncertain. Mutagenesis has previously revealed that the four FtsH homologs encoded by the cyanobacterium Synechocystis sp PCC 6803 are functionally different: FtsH1 and FtsH3 are required for cell viability, whereas FtsH2 and FtsH4 are dispensable. To gain insights into FtsH2, which is involved in selective D1 protein degradation during PSII repair, we used a strain of Synechocystis 6803 expressing a glutathione S-transferase (GST)-tagged derivative (FtsH2-GST) to isolate FtsH2-containing complexes. Biochemical analysis revealed that FtsH2-GST forms a hetero-oligomeric complex with FtsH3. FtsH2 also interacts with FtsH3 in the wild-type strain, and a mutant depleted in FtsH3, like ftsH2 2 mutants, displays impaired D1 degradation. FtsH3 also forms a separate heterocomplex with FtsH1, thus explaining why FtsH3 is more important than FtsH2 for cell viability. We investigated the structure of the isolated FtsH2-GST/FtsH3 complex using transmission electron microscopy and single-particle analysis. The three-dimensional structural model obtained at a resolution of 26 Å revealed that the complex is hexameric and consists of alternating FtsH2/FtsH3 subunits.

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Members of the DegP/HtrA (or Deg) family of proteases are found widely in nature and play an important role in the proteolysis of misfolded and damaged proteins. As yet, their physiological role in oxygenic photosynthetic organisms is unclear, although it has been widely speculated that they participate in the degradation of the photodamaged D1 subunit in the photosystem two complex (PSII) repair cycle, which is needed to maintain PSII activity in both cyanobacteria and chloroplasts. We have examined the role of the three Deg proteases found in the cyanobacterium Synechocystis sp. PCC 6803 through analysis of double and triple insertion mutants. We have discovered that these proteases show overlap in function and are involved in a number of key physiological responses ranging from protection against light and heat stresses to phototaxis. In previous work, we concluded that the Deg proteases played either a direct or an indirect role in PSII repair in a glucose-tolerant version of An inevitable consequence of the light reactions of oxygenic photosynthesis is the formation of highly reactive molecules, such as reactive oxygen species (ROS) 5 and amino acid free radicals, which can cause irreversible damage to a variety of cellular components including nucleic acids, lipids, pigments, and proteins (1, 2). The photosystem two complex (PSII), which functions as the light-driven water:plastoquinone oxidoreductase in oxygenic photosynthetic electron transport, is particularly prone to light-induced damage (3). Of the more than 25 protein subunits found in PSII, the D1 reaction center subunit appears to be the major target for photodamage (4 -6). To maintain activity, a damaged PSII complex is repaired through the specific replacement of the damaged subunit (usually D1) by a newly synthesized subunit (3). Despite the importance of the PSII repair cycle for maintaining optimal photosynthetic rates in vivo, the molecular details of this repair process remain unclear.Recently, attention has focused on the identity of the proteases that are involved in removing damaged D1 from the PSII complex. In the case of chloroplasts, in vitro experiments suggest that D1 degradation occurs in a two-step process involving the participation of two classes of protease (7). First, a member of the DegP/HtrA family of proteases (or Deg proteases), originally designated DegP2 but now renamed Deg2 (8), is thought to cleave damaged D1 between trans-membrane helices four and five on the stromal side of the membrane to generate N-terminal and C-terminal fragments of ϳ23 and 10 kDa, respectively. Subsequently, the 23-kDa fragment, and possibly the 10-kDa fragment, is removed from the membrane by one or more members of the FtsH protease family (9).Selective D1 degradation also occurs in cyanobacteria such as Synechocystis sp. PCC 6803 (10). Analysis of the genome sequence of Synechocystis sp. PCC 6803 has identified three members of the Deg proteases and four members of the FtsH family of proteases (11). Mutagenesis experiments have so far demonstr...